[Experimental Studies On The Efficacy Of Thiabendazole Against The Migratory Stages Of Ascarids In Mouse]
INTRODUCTION: It is known that the larvae of ascarids have migrating phase before they reach the intestine. Stewart (1916) reported the pulmonary migration of ascaris larvae in normal host. Beaver et al. (1952) demonstrated the ascaris larvae of animal origin from the biopsied human liver, and applied the term "visceral larva migrans" to the migration of larval nematodes in unsuitable hosts. Either in normal or abnormal host, the migrated larvae cause inflammatory changes in the tissues and produce corresponding symptoms. There have been a considerable number of anthelminthics for the ascaris adult worm, but very few reports concerning the migrating larvae. Smirnov (1932) found no larvicidal effect of santonin and chenopodium oil on the migrating phase. Snyder (1961) also reported that diethylcarbamazine did not relieve the symptoms of visceral larva migrans. Recently, thiabendazole has appeared as a broad spectrum anthelminthic and Brown (1961) reported that the chemical inhibited the development of helminth larvae affecting the migratory phases of roundworms and kidney worms in swine. The present study was designed to confirm the previous reports concerning the anthelminthic effect of thiabendazole and to examine the mechanism of its activity. MATERIALS: Animal: White mice, weighing 18-26 gms, were used regardliss of sex. Parasites: Eggs from the 3cm distal portion of uteri of Toxocara canis and Ascaris lumbricoides were sampled and cultured in 0.5% formalin solution under room temperature for 40-50 days. The embryonated eggs were used for the experiment. Virus: Infuenza A/swine/1957/12 N.I.H., U.S.A. December 20, 1965. Chemical: Thiabendazole; 2-4'-thiazolyle)-benzimidazole, Merck Sharp and Dohme Co. 50% aqueous suspension of the chemical was used for experiment. METHODS: White mice were infected each orally using the stomach tube with 500 eggs of canine-ascaris or 800-1,000 eggs of human ascaris according to the experimental purposes. The viral infection was done by inhalation of 2-3 drops of emulsion containing viurs, and the drug was given by stomach tube. The average dose was 250 mg/kg. Recovery of larvae from the tissues: The larvae in the brain were examined under the microscope by pressing the tissue between two slides. The tissues of liver, lung, and carcasses were macerated with Waring blendor. The macerated tissue was suspended in 20 cc freshly prepared artificial gastric juice (pepsin 1 gm, HCL 0.5 cc, NaCL 0.85 gm, distilled water 100 cc), and incubated over night at 37 degrees C. The sample was centrifuged and the sediments examined for larvae. Inthe first experiment, the fate of the migrating larvae after drug administration was determined in early observation group and late observation group. The early observation group: Three days after the infection of 500 eggs of Toxocara canis, 30 of the mice were diviede into two subgroups; having had a single dose and three doses of drug respectively. Four days after the first dose, the mice were sacrificed and the larvae in tissue were examined. The late observation group: The procedure was the same. The mice were sacrificed 14th day after drug administration. In the second experiment, 30 mice infected with 1,000 eggs of human ascaris each were divided into three groups. One group was control and two groups were group of drug administration. In one group the drug was given two days after the infection and the other in 6 days. Allthe mice were sacrificed on the 8th day after the infection. In the third experiment, 45 mice which were infected with 800 eggs of human ascaris each were divided into three groups. In one group the drug was given 24 hours before the infection and in the other 24 hours after the infection and control A mouse of each group was sacrificed every day for 15 days. In the fourth experiment 100 mice were divided into five groups, I, II, III, IV, V. Group I was infected with influenza virus only, and group II was infeted with 800 eggs of human ascaris only. Group III was infected with the influenza virus 7 days after the ascaris infection. Group IV was treated with a single dose of the drug 24 hours before the infection and the virus was infected 7 days later. Group V was treated with the same dose of drug 24 hours after the infection and virus was infected 7 days later. The fatality of each group was observed every day and also the pathological changes of the lungs in each mouse were examined. RESULT: 1)The number of larvae in the tissues of mice treated with thiabendazole was different according to the observation period and the the number of drug administration. In the early observation group: The number of larvae in the single dose group was 40.9 ± 2.25 (mean ± standard error) The number in organs were 12.8 ± 1.69 in brain, 19.6 ± 1.51 in liver and 8.5 ± 0.88 in muscle. The number in the three doses group was 35.6 ± 1.64. The number in organs were 9.6 ± 0.87 in brain, 16.8 ± 1.75 in liver and 9.2 ± 0.82 in muscle. The average number of larvae from control mice was 85.7 ± 7.45 and the average numbers from different parts of tissue; brain, liver and muscle were 19.7 ± 1.93, 50.8 ± 7.23 and 15.2 ± 1.38 respectively. The average numbers of larvae of single dose group and three dose group were reduced in proportion of 52.2% and 58.5% respectively compared with that of control group. In the late observation group the number of larvae from the single dose group was 28.9 ± 1.35. The numbers in organs were 8.6 ± 0.42 in brain, 10.8 ± 1.13 in liver and 9.5 ± 0.87 in muscle. The number from the three doses group was 26.1 ± 1.01, and the numbers in tissue were 11.3 ± 0.72 in brain, 7.8 ± 0.70 in liver and 19.6 ± 1.45 in muscle. The reduction rates in single dose group and three doses group were 59.3% and 63.2% respectively compared with control group. 2) The numbers of the larvae were examined according to the time of drug administration. Tn the earlier group which were given 3 days after the infection, the numbers of the larvae were 3.5 ± 0.66 in liver and 8.7 ± 0.93 in lungs. But in the later group, the numbers were 7.4 ± 1.04 in liver and 14.4 ± 1.39 in lungs. In the control group, the numbers were 8.7 ± 0.94 in liver and 31.9 ± 1.48 in lungs. The reduction rate of the numbers of larvae from liver and lungs in the early drug administration group were 59.7% and 72.9% respectively. In the delayed drug administrating group, 14.9% and 54.9% were reduced in liver and lungs respectively compared with those of control group. 3) The numbers of the larvae in tissues were different according to the method of drug administration; the groups of durg administration before the infection and after the infection, and control group were 30.6 ± 4.71, 35.9 ± 4.86 and 52.0 ± 6.73 respectively. The numbers of recovered larvae in the mice of drug given before and after infection were reduced to 42.3% and 31.1% as compared with the control group. The peak number of recoverd larvae was observed on the 7th to 8th day in the control group and the group of mice of drug administration before the infection, but on the group of mice of drug administration after the infection was appeared on the 8th to 10th day. The numbers of larvae from liver in the group of mice of drug administration before and after the infection, and control were 16.4 ± 2.93, 19.9 ± 3.16 and 25.8 ± 4.02, respectively. The peak of the number in the liver appeared on the 9th day in the group of drug administration before infection, and in 10th day in the group of drug administration after infection, but in the control group the peak appeared on the 4th day after infection. The numbers of larvae from lungs were 71 ± 1.54, 9.1 ± 1.62 and 12.9 ± 2.42 in the group of before, after and control respectively. The reduction rates were 44.9% and 29.4% as compared with control group. The peak of the number of recovered larvae was shown on the 9th day in the group before infection, in 10th day in the group after infection but in control group the peak appeared on the 8th and 9th day. The larvae in intestinal contents of the group of drug administration before and after infection were reduced 65.6% and 54.7% respectively as compared with control group. 4) The drug also effected the life span of the experimental animals. The group of ascaris infection alone showed the longest period of 13.1 ± 0.90 days. The group was infected by virus alone showed 9.9 ± 0.80 days. The group which was infected by ascaris and virus showed the shortest 3.8 ± 0.40 days. However the groups IV and V which were treated with the drug before and after the infection had almost two times longer longivity than the combined infection group. The pathological findings of the lungs were also different according to the drug administration. The ascaris only group showed the light edematous changes and hemorrhagic spots. The viral group showed severe inflammation and edematous changes on whole lungs and the combined group showed severe inflammation and edema with massive hemorrhage on entire lung field. However the treated group showed much lighter changes than in the group of combined infection. CONCLUSION: The following results were obtained in the present study concerning the effectiveness of thiabendazole upon the larvae of the migrating stages. 1) In the early observation group: The average number of larvae of the group treated with single dose and the group treated with three doses were reduced in proportion of 52.2%, 58.5 % respectively compared with control group. 2) In the late observation group: The reduction rate in the group treated with singel dose and group treated with three doses were 59.3 % and 63.2 % respectively compared with control group. 3) The reduction rates of larvae from liver and lungs in the early drug administration group were 72.9 % and 59.7 % respectively, and 14.9 % and 54.8 % in the delayed drug administration group. 4) In the group of drug given before and after infection, the number of recovered larvae were reduced 42.2 % and 31.1 % respectively compared with the control group. 5) The peak number in organs was delayed 1 to 2 days in the treated group than that of control group. 6) The survival period of the infected mouse was prolonged by the drug administration. 7) The pathological changes were reduced by the administration of the drug. Through above results, it was concluded that thiabendazole reduced the number of migrating larvae and delayed the normal migration of the larvae in tissues and reduced the pathological changes in the tissues.